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Freeze-Etching Electron Microscopy: Recent Developments and Application to the Study of Biological Membranes and Their Components

  • T. Gulik-Krzywicki
Part of the NATO Advanced Science Institutes Series book series (NSSA, volume 71)

Abstract

Electron microscopy, which is one of the most direct and most powerful techniques for obtaining structural information, has been applied only sparingly in the study of untreated samples. Examination of such samples is virtually impossible due to the damaging effects of the high vacuum and electron beam and because of the very weak contrast provided by the biological molecules. Generally pretreatments (chemical fixation and staining) to permit electron microscopic observation may modify the sample structure.1 One procedure which seems to avoid some of the pitfalls of the other techniques is cryofixation. Frozen hydrated samples may thus be examined directly2 or by freeze fracture electron microscopy. Ideally, when cryofixation is used, the sample should be quenched rapidly enough to avoid structural alterations due to temperature induced structural transitions, change of the partial specific volumes, ice crystal formation, etc. Practically, this goal may be difficult to achieve and one must assess the extent of preservation of the sample structure after cryofixation. Among the methods that might be used for such an assessment, X-ray, neutron or electron diffraction, which provide detailed structural information, are probably the most straightforward. We have developed an approach based on the combined use of ambient and low temperature X-ray diffraction and freeze-etching electron microscopy to study the effect of quenching upon sample structure. This approach was applied first to study a variety of lipid-wate phases of known structure cryofixed by different procedures.3–4 More recently the same approach was used to study solutions of low density serum lipoproteins5. The results of these studies provided some understanding of the factors which affect sample structure during quenching and permitted the elaboration of a more general approach for structural investigations by freeze etching electron microscopy. In this paper we will describe this general approach and its use in the study of biological membrane components. The use of freeze-etching electron microscropy for the study of native membranes will be illustrated by some recent studies of the molecular mechanism of synaptic transmission. In the latter case, the use of ultrarapid cryofixation procedures allow one to follow rapid morphological changes taking place within the presynaptic membrane after different types of stimulation which lead to the release of acetylcholine.

Keywords

Biological Membrane Fracture Plane Sample Structure Partial Specific Volume Lipid Hydrocarbon Chain 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Plenum Press, New York 1985

Authors and Affiliations

  • T. Gulik-Krzywicki
    • 1
  1. 1.Centre de Génétique MoléculaireC.N.R.S.Gif sur YvetteFrance

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